Target (protein/gene name): adenylosuccinate synthetase *NCBI Gene # or RefSeq#: NCBI Gene ID:814251 *Protein ID (NP or XP #) or Wolbachia#: XP_001350257.1 *Organism (including strain):P. falciparum 3D7 Etiologic Risk Group (see link below): India, Africa, and Some Areas in South America and is often seen in pregnant women, children under 5 years, people with HIV/AIDS and non-immune travelers. http://www.cdc.gov/malaria/map/ Disease Information(sort of like the Intro to your MiniResearch Writeup: Malaria is caused by a parasite Plasmodium falciparum. The protozoan parasite is infects the humans thru a vector of mostly mosquitos. Malaria symptoms include fever, chills, and flu-like illness. When untreated the disease can worsen leading to complication and even death. http://www.cdc.gov/malaria/ http://www.niaid.nih.gov/topics/Malaria/understandingMalaria/Pages/cause.aspx
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 1P9B Current Inhibitors: The presence, or over abundance of AMP, GMP, GDP and adenylosuccinate acts as a feedback inhibitor http://www.proteopedia.org/wiki/index.php/Adenylosuccinate_Synthetase
Expression Information (has it been expressed in bacterial cells):partially purified with E. coli- homogeneous preparation of the E. coli enzyme using ammonium sulfate fractionation and DEAE-cellulose and Sephadex G-100 chromatography. Purification Method:Ornstein and Davis (16), with 7% polyacrylamidegels, pH 8.3 Tris-glycine buffer and 20 to 100 pg of protein. http://www.jbc.org/content/249/2/459.full.pdf+html
Image of protein (PyMol with features delineated and shown separately): Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MAIFDHQIKN VDKGNVVAIL GAQWGDEGKG KIIDMLSEYS DITCRFNGGA NAGHTISVNDKKYALHLLPC GVLYDNNISV LGNGMVIHVK SLMEEIESVG GKLLDRLYLSNKAHILFDIH QIIDSIQETK KLKEGKQIGT TKRGIGPCYS TKASRIGIRL GTLKNFENFK NMYSKLIDHL MDLYNITEYD KEKELNLFYN YHIKLRDRIV DVISFMNTNL ENNKKVLIEG ANAAMLDIDF GTYPYVTSSC TTVGGVFSGL GIHHKKLNLV VGVVKSYLTR VGCGPFLTEL NNDVGQYLRE KGHEYGTTTK RPRRCGWLDI PMLLYVKCIN SIDMINLTKL DVLSGLEEIL LCVNFKNKKT GELLEKGCYP VEEEISEEYE PVYEKFSGWK EDISTCNEFD ELPENAKKYI LAIEKYLKTP IVWIGVGPNR KNMIVKKNFN LN
*length of your protein in Amino Acids: 442 Amino Acids Molecular Weightof your protein in kiloDaltons using the**Expasy ProtParam**website:50.0218 kDA MolarExtinction coefficientof your protein at 280 nm wavelength:1.078
Do Not Need this info forSpring(but still copy these lines to your Target page for now) *CDS Gene Sequence (codon optimized) - copy fromoutputofPrimer DesignProtocol (paste as text only): *GC% Content for gene (codon optimized): Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works outputtext file-that should be saved in yourGoogle Docsfolder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file ifyou are notsure how to. Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: NCBI Gene ID:814251
*Protein ID (NP or XP #) or Wolbachia#: XP_001350257.1
*Organism (including strain): P. falciparum 3D7
Etiologic Risk Group (see link below): India, Africa, and Some Areas in South America and is often seen in pregnant women, children under 5 years, people with HIV/AIDS and non-immune travelers.
http://www.cdc.gov/malaria/map/
Disease Information(sort of like the Intro to your MiniResearch Writeup: Malaria is caused by a parasite Plasmodium falciparum. The protozoan parasite is infects the humans thru a vector of mostly mosquitos. Malaria symptoms include fever, chills, and flu-like illness. When untreated the disease can worsen leading to complication and even death.
http://www.cdc.gov/malaria/
http://www.niaid.nih.gov/topics/Malaria/understandingMalaria/Pages/cause.aspx
Link to TDR Targets page (if present): http://www.tdrtargets.org/targets/view?gene_id=2266
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/gene/814251
https://www.patricbrc.org/portal/portal/patric/Taxon?cType=taxon&cId=262
Essentiality of this protein:
- “catalysing the first committed step in the synthesis of AMP from IMP”
- “in regulation of protein function” http://www.sciencedirect.com/science/article/pii/S0022283603014426#
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):MonomerComplex of proteins: Simple only one chain
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):0.8 from TDR Targets
*EC#: 6.3.4.4
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.4.4#
NATURAL SUBSTRATES
Enzyme Assayinformation (spectrophotometric, coupled assay ?, reagents): Spectrophotometric
-- link (or citation) to paper that contains assay information: “AMP deaminase (EC 3.5.4.6) [33] ; adenylosuccinate synthetase (EC 6.3.4.4) [34] ; adenylosuccinate lyase (EC 4.3.2.2) [35] ; IMP dehydrogenase (EC 1.2.1.14)”
http://ac.els-cdn.com/0166685182900354/1-s2.0-0166685182900354-main.pdf?_tid=59650bd0-e486-11e4-9102-00000aacb35f&acdnat=1429222820_2b56697db3d5e9cb7c8030a9e56e1743
--links to assay reagents (substrates) pages.http://www.jbc.org/content/249/2/459.full.pdf+html
--- List cost and quantity of substrate reagents, supplier, and catalog #:
HEPES Buffer: BP299-100 on Fisher $83.11 for 100mL
IMP: AC22626-0050 on Fisher 28.26 for 5g
GTP: ICN10071010 on Fisher 32.16 for 10g
MgCl2: BP6114 on Fisher for $14.70 for 1mL, 25mM
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1P9B
Current Inhibitors: The presence, or over abundance of AMP, GMP, GDP and adenylosuccinate acts as a feedback inhibitor http://www.proteopedia.org/wiki/index.php/Adenylosuccinate_Synthetase
Expression Information (has it been expressed in bacterial cells):partially purified with E. coli- homogeneous preparation of the E. coli enzyme using ammonium sulfate fractionation and DEAE-cellulose and Sephadex G-100 chromatography.
Purification Method:Ornstein and Davis (16), with 7% polyacrylamidegels, pH 8.3 Tris-glycine buffer and 20 to 100 pg of protein.
http://www.jbc.org/content/249/2/459.full.pdf+html
Image of protein (PyMol with features delineated and shown separately):
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MAIFDHQIKN VDKGNVVAIL GAQWGDEGKG KIIDMLSEYS DITCRFNGGA
NAGHTISVND KKYALHLLPC GVLYDNNISV LGNGMVIHVK SLMEEIESVG
GKLLDRLYLS NKAHILFDIH QIIDSIQETK KLKEGKQIGT TKRGIGPCYS
TKASRIGIRL GTLKNFENFK NMYSKLIDHL MDLYNITEYD KEKELNLFYN
YHIKLRDRIV DVISFMNTNL ENNKKVLIEG ANAAMLDIDF GTYPYVTSSC
TTVGGVFSGL GIHHKKLNLV VGVVKSYLTR VGCGPFLTEL NNDVGQYLRE
KGHEYGTTTK RPRRCGWLDI PMLLYVKCIN SIDMINLTKL DVLSGLEEIL
LCVNFKNKKT GELLEKGCYP VEEEISEEYE PVYEKFSGWK EDISTCNEFD
ELPENAKKYI LAIEKYLKTP IVWIGVGPNR KNMIVKKNFN LN
*length of your protein in Amino Acids: 442 Amino Acids
Molecular Weightof your protein in kiloDaltons using the**Expasy ProtParam**website: 50.0218 kDA
MolarExtinction coefficientof your protein at 280 nm wavelength:1.078
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
atggcgatttttgatcatcagattaaaaacgtggataaaggcaacgtggtggcgattctg ggcgcgcagtggggcgatgaaggcaaaggcaaaattattgatatgctgagcgaatatagc gatattacctgccgctttaacggcggcgcgaacgcgggccataccattagcgtgaacgat aaaaaatatgcgctgcatctgctgccgtgcggcgtgctgtatgataacaacattagcgtg ctgggcaacggcatggtgattcatgtgaaaagcctgatggaagaaattgaaagcgtgggc ggcaaactgctggatcgcctgtatctgagcaacaaagcgcatattctgtttgatattcat cagattattgatagcattcaggaaaccaaaaaactgaaagaaggcaaacagattggcacc accaaacgcggcattggcccgtgctatagcaccaaagcgagccgcattggcattcgcctg ggcaccctgaaaaactttgaaaactttaaaaacatgtatagcaaactgattgatcatctg atggatctgtataacattaccgaatatgataaagaaaaagaactgaacctgttttataac tatcatattaaactgcgcgatcgcattgtggatgtgattagctttatgaacaccaacctg gaaaacaacaaaaaagtgctgattgaaggcgcgaacgcggcgatgctggatattgatttt ggcacctatccgtatgtgaccagcagctgcaccaccgtgggcggcgtgtttagcggcctg ggcattcatcataaaaaactgaacctggtggtgggcgtggtgaaaagctatctgacccgc gtgggctgcggcccgtttctgaccgaactgaacaacgatgtgggccagtatctgcgcgaa aaaggccatgaatatggcaccaccaccaaacgcccgcgccgctgcggctggctggatatt ccgatgctgctgtatgtgaaatgcattaacagcattgatatgattaacctgaccaaactg gatgtgctgagcggcctggaagaaattctgctgtgcgtgaactttaaaaacaaaaaaacc ggcgaactgctggaaaaaggctgctatccggtggaagaagaaattagcgaagaatatgaa ccggtgtatgaaaaatttagcggctggaaagaagatattagcacctgcaacgaatttgat gaactgccggaaaacgcgaaaaaatatattctggcgattgaaaaatatctgaaaaccccg attgtgtggattggcgtgggcccgaaccgcaaaaacatgattgtgaaaaaaaactttaac ctgaac
*GC% Content for gene:41%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
*CDS Gene Sequence (codon optimized) - copy fromoutputofPrimer DesignProtocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works outputtext file-that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):